| The solid-phase attraction
For an increasing array of applications, researchers find SPE both adjunct and alternative to liquid–liquid chromatography.
 Solid-phase extraction (SPE) is simply, at base, a form of solid adsorption chromatography, ranging in complexity from simple hydrophobic or hydrophilic partitioning to ion-exchange or affinity chromatography. A sample is loaded onto a column containing a solid particulate medium chosen for its specific adsorption qualities; the desired components in the sample bind noncovalently to the solid medium while impurities flush through. The medium can then be washed free of the contaminants and the desired materials eluted by an appropriate change in solvent. The more the particulate medium is designed to bind to a narrow class of compounds, the closer to affinity chromatography the procedure approaches. In fact, the greatest excitement currently in SPE is the development of molecularly imprinted polymers (MIPs), which truly transform traditional SPE into a specific affinity technique.
Sorbents for SPE are packaged commercially in three basic formats: solid disks, prepared cartridges, and a variety of standard syringe barrels (typically polypropylene barrels rather than glass, which is primarily used in conjunction with Teflon couplings to eliminate interference from contaminating plasticizers). From the first use of silica, alumina, Florosil, and kieselguhr in the 1930s as solid adsorbant, to the introduction of the Sep-Pak by Waters in 1978, to the coining of the term “SPE” in 1982 by employees of the J. T. Baker Co., to the present, the use of these “trace enrichment” techniques has proved critical to the development of modern chemistry (1).
Applications of SPE run the gamut from simple cleanup using relatively common sorbents for a broad variety of compound classes in sample prep and concentration before standard chromatography to highly specific extraction of particular molecules in biomedicine and pollution studies. SPE media can be used in a variety of forms, from loose powders to solid cylinders to membrane formats—but selective adsorption and release of desired compounds remains at the heart of each design.
Aiding Chromatography
Because traditional SPE is somewhat nonspecific, binding classes of compounds rather than unique molecules, it has most often been used as a preliminary sample purification and concentration step for large-scale liquid–liquid chromatography or as a first step before analysis using a variety of spectroscopic techniques. From this perspective, SPE is merely one of several highly useful methods for sample preparation—an extremely critical although somewhat narrow role.
Environmental analysis is a typical application of SPE for trace enrichment before a more rigorous and informative GC or LC analysis. Because of EPA regulations in the United States and a variety of EU statutes in Europe, water samples can range from 100 mL to 1 L in size. And large numbers of samples can be required for coverage of a region or problem site. Obviously, some form of concentration is required. Such volumes can easily be passed through an SPE cartridge or disk containing the proper sorbent for the compounds of interest. These can then be eluted in a much smaller volume of solvent for specific identification of pollutants through injection into a GC or LC. One of the benefits of such filtration–concentration is that it can be done either in-line or off-line and also lends itself to automated analysis.
Replacing Chromatography
More and more today, researchers are working to transform SPE into a replacement for standard liquid–liquid chromatography by developing different solid-phase chemistries to expand the repertoire or hone the specificity of the technique. In fact, the sorbents used in typical SPE are often identical to those used for liquid chromatography. They can be used in typical normal phase, reversed phase, ion exchange, or size exclusion (wide-pore) extraction. These different categories of sorbents are typically produced by chemically bonding specific functional groups to silica gel or a variety of polymeric beads or ion-exchange media (a typical particle is 40 µm in diameter with 60 Å pores). Wercinski (2) presents an extensive table and discussion of sorbents for each type of traditional SPE as well as a discussion of the general applications used. Reversed-phase setups, with a hydrophobic solid phase, are important for such applications as examining drugs in bodily fluids, testing for pesticides in water, and desalting peptides. Normal phase applications (with a polar solid phase) include lipid classification, pesticide analysis, and the separation of plant pigments.
Moving to Membranes
Although traditional SPE evolved in the form of cartridges or particulates in syringes, all of the advantages of membrane filtration can be adapted to SPE by embedding the solid-phase extraction particles into a flat matrix. According to 3M, one of many companies producing SPE products (see box, “Some Companies Involved in SPE and SPME”), the use of such membranes “results in a denser more uniform extraction medium than can be achieved in traditional solid-phase extraction.” Especially useful is the fact that such disk membranes can greatly increase extraction efficiency and reduce the solvent and sample volumes that can be analyzed, in addition to requiring significantly smaller elution volumes, and thus resulting in improved concentration efficiencies of the purified product.
But the true utility of SPE lies in the fact that it can be designed to fit whatever volumes or architectures a particular chemical system requires—from the filaments in micro-extraction to the miniature membrane disks used to automate SPE in the bottom of 96-well plates for a wide variety of combinatorial chemistry applications. (Of course, if SPE is used in large volumes in a full-sized column, it is simply referred to as adsorption chromatography!) Double-disk solid-phase extraction has been developed in which two separate membrane matrices with different properties are used together to enhance the trace enrichment process of particular chemicals. A good example of this is provided by Ferrer et al. (3), who used two disks to simultaneously clean and enrich herbicides and metabolites from environmental samples. The first membrane bound natural organics that normally interfered with analysis, and the second bound the majority of the now-available herbicides from the water. Differential alcohol elution released the compounds of interest and retained the natural organics on the membrane.
Antibodies et al.
A wide variety of functionalized polymeric sorbents and highly cross-linked polymers have been developed as new solid phases for use in modern SPE. According to Wercinski (2), some of the most important of these include graphitized carbon, functionalized styrene-divinylbenzene, and a variety of restricted-access sorbents. Of particular note has been the increasing value of affinity SPE, especially using immunosorbent platforms.
Most chemists are aware of the utility of standard affinity chromatography for purification purposes. But as with all of the other forms of liquid chromatography, SPE has found a place for antibody techniques as well. As expected, antibodies to the analyte in question are bound to the silica or polymeric matrix used for extraction. The analyte, behaving like a typical antigen, then binds in a lock-and-key fashion to the specific antibody sites. An appropriate buffer wash can release the concentrated molecules from the matrix, whether it be in syringe, cartridge, or membrane. Originally, antibody technology was poorly adaptable to small organic molecules because the body would not recognize them antigenically. But new methods of antigen presentation and the development of monoclonals allowed for the production of highly specific antibodies to a wide variety of industrially and medically important small molecules.
Play MISPE for Me
According to Masque et al. (4), the recent popularity of using immunosorbants for SPE of analytes from complex matrices such as blood, urine, and environmental water samples has led to a demand for less difficult to develop and more cost-effective alternatives to antibodies.
Synthetic antibody mimics, known as molecular imprinted polymers or MIPs, seem to provide the answer. MIPs are “cross-linked molecules bearing ‘tailor-made’ binding sites for target molecules” (4). They are formed by an ingenious method of complexing monomers containing functional groups with a target compound. The monomers bind to appropriate locations on the target. A large excess of cross-linker is subsequently added to form “a highly cross-linked polymer network” from which the target (which has now served as a template) is then removed by washing or digestion. The process is the equivalent to forming a mold that “fits” specific portions of the target molecules in the same sort of lock-and-key fashion as would an antibody or enzyme. The new molecularly imprinted solid-phase extraction (MISPE) template can then be used to selectively bind new target molecules, even in the presence of complex mixtures. MIP technology provides the ability to create the equivalent of matrix-embedded synthetic antibodies on demand for use in a wide variety of concentration schemes. Like antibodies, individual MIPs are designed to bind to a very narrow specificity of target—the one to which they were initially formed, or very close variations on that pattern. Masque and his colleagues demonstrated how MISPE can be adapted to environmental applications. They synthesized an MIP to 4-nitrophenol and found that they could selectively preconcentrate this pollutant from a river water solution spiked with 10 other EPA priority phenolic compounds at microgram-per-liter levels.
An alternative to MISPE is molecular recognition (MR) technology. In this case, instead of adding standard functional groups to silica or polymeric beads, a synthetic ligand designed specifically to bind to known portions of the target molecule is used. This allows for a typical affinity chromatography approach, and avoids some of the costs and complications often associated with antibodies (not least of which is the benefit from both MISPE and MRSPE of not requiring some level of biological or at least biochemical expertise).
Moving to Micro
The ability to scale down SPE has become critical in an era in which increased efficiencies are a top priority. In fact, SPME (the M standing for micro) has become a much discussed spinoff of traditional SPE for dealing with high-value, low-volume samples. According to Wercinski (2), SPME has only become commercially available since the mid-1990s. Rather than relying on loose particles or membranes, the technique uses a small, solid rod of fused silica (typically 1 cm long with a 0.11 cm outer diameter). The silica is precoated with an absorbent polymer chosen for the particular application required. For example, polar coatings such as polyacrylate and polyethylene glycol are used to bind polar compounds; nonpolar coatings (such as polydimethylsiloxane) are used for nonpolar compounds. Key benefits of using such a fiber include its ability to be inserted into a sample for drawing up material and its structural strength, which allows it to be moved throughout a sample to prevent the formation of a depletion zone around the fiber. Applications of SPME are innumerable and have been the subject of several reviews (2, 5).
One of the great benefits of SPME is its efficient ability to sample and bind vaporized compounds, allowing it to be used to sample gases for particular compounds. In such cases, the bound sample can be desorbed directly into a GC injector for analysis.
Coming Extractions
The future of SPE seems secure. Already a mainstay of environmental analysis, it has recently been adapted to the stringent requirements of modern biotechnology automation upon being melded with the ubiquitous 96-well plate by a variety of companies. Researchers Peng et al. (6) showed that such a well-plate system could make a valuable front-end purification/concentration step for LC-MS-MS used for the determination of protease inhibitors in plasma and cartilage samples. The technique demonstrated significant improvements in time-saving and in the sensitivity of the MS detection.
SPE has even found a place on microchips. Recently, monolithic porous polymers produced by photoinitiated polymerization have been developed for use in on-chip extraction and preconcentration within the channels of a microfluidic device (7). In addition, SPE has proven its worth in that it can now be routinely automated for cost effectiveness and efficiency for everything from drug applications (from discovery to cocaine abuse monitoring) to environmental applications (from monitoring pesticides to identifying industrial waste). So it is obvious that whatever the future holds in improvements of analysis, SPE will accompany each new development, providing its time-honored benefits of analyte purification and concentration with ever more precision and technical ease.
References
- Thurman, E. M.; Mills, M. S. Solid-Phase Extraction: Principles and Practice; Wiley & Sons: New York, 1998.
- Wercinski, S. A. S., Ed. Solid Phase Microextraction: A Practical Guide; Marcel Decker: New York, 1999.
- Ferrer, I.; et al. Anal Chem. 1999, 71, 1009–1015.
- Masgue, N.; et al. Anal. Chem. 2000, 72, 4122–4126.
- Pawliszyn, J., Ed. Applications of Solid Phase Microextraction; The Royal Society of Chemistry: Cambridge, 1999.
- Peng, S. X.; et al. Anal. Chem. 2000, 72, 1913–1917.
- Yu, C.; Davey, M. H.; Svec, F.; Frechet, J. M. J. Anal. Chem. 2001, 73, 5088–5096.
Mark S. Lesney is a senior editor and Cullen T. Vogelson is a former assistant editor of Today’s Chemist at Work. Currently, Vogelson is a senior research scientist with Alcon Laboratories. Send your comments or questions regarding this article to tcaw@acs.org or the Editorial Office, 1155 16th St N.W., Washington, DC 20036.
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